Calcium Imaging of Transfected Tas2r Receptors

HEK-293 cells were transfected with Tas2r105- or Tas2r108-coding plasmids using Lipofectamine 2000, according to the manufacturer’s protocol. DNA and Lipofectamine were diluted in Opti-MEM and incubated with the cells overnight. Calcium imaging experiments were performed as described previously [18 (link)]. Briefly, coverslips seeded with transfected and non-transfected HEK293 cells were washed three times with Tyrode III buffer containing (in mM) 130 NaCl, 10 HEPES, 10 glucose, 8 CaCl₂, 5 KCl, 10 sodium pyruvate, 5 NaHCO₃, and 1 MgCl₂. Then, cells were loaded with Fura-2-AM (5 µM, Invitrogen) and 0.04% pluronic (Sigma-Aldrich) for 30 min at 37 °C in Tyrode III buffer. Changes in [Ca²⁺]_i levels to denatonium application were studied using an Eclipse FN1 microscope (Nikon Instruments, Amsterdam, The Netherlands). Cells were excited alternately at 340 and 380 nm using Lambda DG-4 high-speed wavelength switcher equipped with a xenon arc lamp (Sutter Instruments, Novato, CA, USA), and emission was detected at 510 nm using an Orca Flash 4.0 camera (Hamamatsu, Herrsching, Germany). Acquired values were processed using the NIS-Elements software (Nikon Instruments, Amsterdam, The Netherlands).